Field
The disclosure is directed to the determination of compounds of interest using microarrays.
Background
High-throughput measurements have begun to provide insight into the intrinsic complexities and dense interconnectivities of biological systems. As examples, whole-genome sequencing has yielded a wealth of information on crucial genes and mutations underlying disease pathophysiology, DNA microarrays have allowed transcription patterns of various cancers to be dissected, and large-scale proteomics methods have facilitated the study of signaling networks in cells responding to various growth factors. However, the ability to rapidly interrogate the sequence-structure-activity relationship of millions of protein variants, with functional read-outs that span a range of biophysical and biochemical measurements, remains a critical unmet need in high-throughput biology. Here, we describe a user-friendly, cost-effective technology we developed to address this need and showcase its capabilities and breadth through novel discovery applications on three distinct protein classes: antibody therapeutics, fluorescent protein biosensors, and enzymes.
Protein engineers rely heavily on directed evolution, a powerful combinatorial screening method which uses selective pressure to evolve proteins with improved properties. Using this approach, libraries are screened to identify proteins with desirable characteristics, such as high affinity binding to a target of interest, stability, expression, or enzymatic activity. Maintaining a genotype-to-phenotype linkage is a fundamental requirement for any directed evolution effort; a protein variant must remain associated with its corresponding DNA sequence to be identified following a screen. This requirement is most easily achieved in assays used to screen for protein binding partners. As examples, genetic fusion of protein variants to microbial cell surface or phage components or translation machinery has allowed rapid identification of target binders from large protein libraries (107-1014 variants) using fluorescence-activated cell sorting (FACS) or panning methods.
Protein analysis methods that employ spatial segregation, such as testing individual enzyme variants in microtiter plates, have expanded protein engineering applications beyond binding interactions, but are generally limited in throughput to 103-105 variants in a typical screen. These relatively small library sizes are restrictive due to the vast theoretical diversity of amino acid search space for a typical protein. Robotic handling systems for assaying protein function in microtiter plates have eased labor, but are still relatively low-throughput (e.g. 100,000 assays per day), and require cost-prohibitive quantities of materials and reagents. Recently, oil-water emulsion droplets created in bulk or combined with microfluidics chips have achieved success in high-throughput enzyme engineering applications, however, this technology can be challenging to implement and does not easily allow temporal measurements of kinetic parameters in real-time during an experiment.